Acute kidney injury (AKI) is a complication of cardiac surgery with short-term morbidity, increased costs of treatment, and poor long-term outcome. Tubular regeneration restores normal tubular architecture and renal function following kidney injury. The goal of the proposed research is to investigate the mechanism of Foxc2 nuclear-cytoplasmic shuttling via during proximal tubule injury and repair. Foxc2 is a member of the Forkhead box (Fox) transcription factors known to be involved in cardiovascular, skeletal, and kidney development, playing a role in specifying mesenchymal cell fates. Upregulation of nuclear Foxc2 in epithelial tumors leads to repression of epithelial markers (E-cadherin and catenins) and an increase in mesenchymal markers (vimentin and -Sma). However, recent evidence from our laboratory has shown that cytoplasmic Foxc2 helps maintain the epithelial state in injured proximal tubule cells. Thus, exporting Foxc2 from the nucleus may serve to moderate the de-differentiation response to acute injury and to promote epithelial re-differentiation during the repair process in proximal tubule cells. My preliminary data suggests that phosphorylation at serine 125 shuttles Foxc2 out of the nucleus in proximal tubule cells. I have also discovered that Foxc2 associates with 14- 3-3 and -actinins in the cytoplasm of mouse proximal tubule cells. My hypothesis that the increase in cytoplasmic Foxc2 that is seen following ischemic tubular injury promotes -actinin stabilization and/or localization to focal adhesions, where it is critical for FA turnover and cell morphogenesis/migration. Specific aim1 determines how Foxc2 nuclear export promotes tubular cell repair by 14-3-3 and -actinin association. Regulatory sites/binding partners will be evaluated by determining the subcellular localization of GFP-tagged Foxc2 constructs in which the putative binding/regulatory site is mutated. Candidate regulatory kinases for these sites will be screened using a phosphoproteomics approach followed by kinase inhibitor and/or knock- down studies to determine regulation of partner proteins with Foxc2 and impact on subcellular (cytoplasmic vs. nuclear) localization. Mutants at two regulatory sites will also be examined to determine if 14-3-3 modulates Foxc2 and -actinin interaction by co-immunoprecipitation. Inhibition of endogenous Foxc2 expression by siRNA transfection will be utilized to examine the effect of endogenous Foxc2 knockdown on -actinin localization. Overexpression of Foxc2 localized to the cytoplasm will determine upregulation of Foxc2 on -actinin and cytoskeleton rearrangements. We will use -actinin and paxillin staining (focal adhesions) viewed and quantified by confocal microscopy. Cell migration will be monitored by wounding/sheet assays. Specific aim2 examines the role of Foxc2 during injury and repair of the proximal tubule in vivo. Knockdown of Foxc2 in vivo by RNAi injection with uptake specifically by the proximal tubule will be performed along with Ischemia/Reperfusion (I/R) to simulate the de-differentiation and re-differentiation process. Western blots will be performed to determine the level of knockdown in the kidney after siRNA of Foxc2. Immunofluorescence by confocal microscopy and western blot analysis of Foxc2, 14-3-3, and -actinin along with paxillin staining for focal adhesions will also be examined. TUNEL staining will be utilized to measure cellular apoptosis. Cumulatively, these aims are designed to better understand the mechanisms of Foxc2 shuttling and the role of Foxc2 in epithelial injury and repair of the proximal tubule. PUBLIC HEALTH RELEVANCE: Ischemia/reperfusion is a major cause of acute kidney injury (AKI) in hospitalized patients and causes complications in cardiac surgery with short-term morbidity, increased costs of treatment, and poor long-term outcome. Tubular regeneration restores normal tubular architecture and renal function following kidney injury and the mechanisms of tubular injury and repair are poorly understood. Extensive examination of the mechanisms and potential therapeutic targets will advance the understanding and potential cure for AKI in proximal tubular regeneration.